This invention relates to lineage-restricted intermediate precursor cells and methods of making and using thereof. More particularly, the invention relates to neuronal-restricted precursors (NRP""s) isolated from mammalian embryos, neuroepithelial stem (NEP) cells, or embryonic stem (ES) cells. These neuronal-restricted precursors are capable of self-renewal and differentiation into neurons, but not into glia, i.e. astrocytes and oligodendrocytes. Methods of generating, isolating, culturing, transfecting, and transplanting such neuronal-restricted precursor cells are also described.
Multipotent cells with the characteristics of stem cells have been identified in several regions of the central nervous system and at several developmental stages. F. H. Gage et al., Isolation, Characterization and Use of Stem Cells from the CNS, 18 Ann. Rev. Neurosci. 159-92 (1995); M. Marvin and R. McKay, Multipotential Stem Cells in the Vertebrate CNS, 3 Semin. Cell. Biol. 401-11 (1992); R. P. Skoff, The Lineages of Neuroglial Cells, 2 The Neuroscientist 335-44 (1996). These cells, often referred to as neuroepithelial stem cells (NEP cells), have the capacity to undergo self renewal and to differentiate into neurons, oligodendrocytes, and astrocytes, thus representing multipotent stem cells. A. A. Davis and S. Temple, A Self-Renewing Multipotential Stem Cell in Embryonic Rat Cerebral Cortex, 362 Nature 363-72 (1994); A. G. Gritti et al., Multipotential Stem Cells from the Adult Mouse Brain Proliferate and Self-Renew in Response to Basic Fibroblast Growth Factor, 16 J. Neurosci. 1091-1100 (1996); B. A. Reynolds et al., A Multipotent EGF-Responsive Striatal Embryonic Progenitor Cell Produces Neurons and Astrocytes, 12 J. Neurosci. 4565-74 (1992); B. A. Reynolds and S. Weiss, Clonal and Population Analyses Demonstrate that an EGF-Responsive Mammalian Embryonic CNS Precursor is a Stem Cell, 175 Developmental Biol. 1-13 (1996); B. P. Williams et al., The Generation of Neurons and Oligodendrocytes from a Common Precursor Cell, 7 Neuron 685-93 (1991).
The nervous system also contains precursor cells with restricted differentiation potentials. T. J. Kilpatrick and P. F. Bartlett, Cloned Multipotential Precursors from the Mouse Cerebrum Require FGF-2, Whereas Glial Restricted Precursors are Stimulated with Either FGF-2 or EGF, 15 J. Neurosci. 3653-61 (1995); J. Price et al., Lineage Analysis in the Vertebrate Nervous System by Retrovirus-Mediated Gene Transfer, 84 Developmental Biol. 156-60 (1987); B. A. Reynolds et al., supra; B. A. Reynolds and S. Weiss, supra; B. Williams, Precursor Cell Types in the Germinal Zone of the Cerebral Cortex, 17 BioEssays 391-93 (1995); B. P. Williams et al., supra. The relationship between multipotent stem cells and lineage restricted precursor cells is still unclear. In principal, lineage restricted cells could be derived from multipotent cells, but this is still a hypothetical possibility in the nervous system with no direct experimental evidence. Further, no method of purifying such precursors from multipotent cells has been described.
As has been shown in copending U.S. patent application Ser. No. 08/852,744, entitled xe2x80x9cGeneration, Characterization, and Isolation of Neuroepithelial Stem Cells and Lineage Restricted Intermediate Precursor,xe2x80x9d filed May 7, 1997, hereby incorporated by reference in its entirety, NEP cells grow on fibronectin and require fibroblast growth factor (FGF) and an as yet uncharacterized component present in chick embryo extract (CEE) to proliferate and maintain an undifferentiated phenotype in culture. The growth requirements of NEP cells are different from neurospheres isolated from E14.5 cortical ventricular zone cells. B. A. Reynolds et al., supra; B. A. Reynolds and S. Weiss, supra; WO 9615226; WO 9615224; WO 9609543; WO 9513364; WO 9416718; WO 9410292; WO 9409119. Neurospheres grow in suspension culture and do not require CEE or FGF, but are dependent on epidermal growth factor (EGF) for survival. FGF itself is not sufficient for long term growth of neurospheres, though FGF may support their growth transiently. NEP cells, however, grow in adherent culture, are FGF dependent, do not express detectable levels of EGF receptors, and are isolated at a stage of embryonic development prior to which it has been possible to isolate neurospheres. Thus, NEP cells may represent a multipotent precursor characteristic of the brain stem and spinal cord, while neurospheres may represent a stem cell more characteristic of the cortex. Nonetheless, NEP cells provide a model system for studying the principles of lineage restriction from multipotent stem cells or precursor cells of the central nervous system. The principles elucidated from the study of NEP cells are expected to be broadly applicable to all CNS precursor cells sufficiently multipotent to generate both neurons and glia. Thus, the present application is intended to be applicable to any CNS precursor cells regardless of their site of derivation as long as they are able to differentiate to both neurons and glial cells.
U.S. Pat. No. 5,589,376, to D. J. Anderson and D. L. Stemple, discloses mammalian neural crest stem cells and methods of isolation and clonal propagation thereof, but fails to disclose cultured NEP cells, cultured lineage restricted precursor cells, and methods of generating, isolating, and culturing thereof. Neural crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), whereas the neuroepithelial stem cells differentiate into neurons and glia of the central nervous system (CNS).
U.S. Ser. No. 08/909,435, filed Jul. 4, 1997, for xe2x80x9cIsolation of Lineage Restricted Neuronal Precursors,xe2x80x9d hereby incorporated by reference in its entirety, describes neuronal restricted precursor (NRP) cells that are capable of differentiating into neurons, but not into glial cells. It was shown that NRP cells can be isolated from NEP cells, as well as directly from embryonic spinal cords.
U.S. Ser. No. 08/980,850, filed Nov. 29, 1997, for xe2x80x9cLineage Restricted Glial Precursors from the Central Nervous System,xe2x80x9d hereby incorporated by reference in its entirety, describes glial restricted precursor (GRP) cells that are capable of differentiating into oligodendrocytes, A2B5+ process-bearing astrocytes, and A2B5xe2x88x92 fibroblast-like astrocytes, but not into neurons. GRP cells can be isolated from differentiating NEP cells, as well as CNS tissue, and differ from oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells in growth factor requirements, morphology, and progeny.
In U.S. patent application Ser. No. 09/073,881, filed May 6, 1998, for xe2x80x9cCommon Neural Progenitor for CNS and PNS,xe2x80x9d hereby incorporated by reference in its entirety, it was shown that NEP cells can be induced to differentiate into neural crest cells as well as other cells of the CNS and PNS.
The neuron-restricted precursor cells described herein are distinct from the NEP cells, GRP cells, neurospheres, and neural crest stem cells that have been described elsewhere. NEP cells are capable of differentiating into neurons or glia whereas NRPs can differentiate into neurons, but not glia, and NEP cells and NRPs display distinct cell markers. GRP cells can differentiate into glia, but not neurons. As mentioned above, neurospheres grow in suspension culture and do not require CEE or FGF, but are dependent on EGF for survival, whereas NRP cells grow in adherent culture and do not express detectable levels of EGF receptors. Further, neural crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), whereas NRP cells differentiate into neurons of the central nervous system (CNS). NRP cells express polysialated or embryonic neural cell adhesion molecule (E-NCAM), but NEP cells, neurospheres, GRP cells, and neural crest cells do not. Therefore, NRP cells are different in their proliferative potential, expression of cell markers, and nutritional requirements from these other cell types.
The ability to isolate and grow mammalian neuronal-restricted precursor cells in vitro allows for of using pure populations of neurons for transplantation, discovery of genes specific to selected stages of development, generation of cell-specific antibodies for therapeutic and diagnostic uses such as for targeted gene therapy, and the like. Further, NRP cells can be used to generate subpopulations of neurons with specific properties, i.e. motoneurons and other neuronal cells for analyzing neurotransmitter functions and small molecules in high throughput assays.
Moreover, the methods of obtaining NRP cells from NEP cells or embryonic stem (ES) cells provides for a ready source of a large number of post-mitotic neurons. Post-mitotic cells obtained from a tumor cell line are already being commercially marketed (e.g., Clontech, Palo Alto, Calif.). The present invention is also necessary to understand how multipotent neuroepithelial stem cells become restricted to the various neuroepithelial derivatives. In particular, culture conditions that allow the growth and self-renewal of mammalian neuronal-restricted precursor cells are desirable so that the particulars of the development of these mammalian stem cells can be ascertained. This is desirable because a number of tumors of neuroepithelial derivatives exist in mammals, particularly humans. Knowledge of mammalian neuroepithelial stem cell development is therefore needed to understand these disorders in humans.
In view of the foregoing, it will be appreciated that isolated populations of mammalian lineage restricted neuronal precursor cells and methods of generating, isolating, culturing, transfecting, and transplanting such cells would be significant advancements in the art.
It is an object of the present invention to provide isolated (pure) populations of mammalian neuronal-restricted precursor cells and their progeny.
It is another object of the invention to provide methods of generating, isolating, culturing, and regenerating of mammalian lineage-restricted neuronal precursor cells and their progeny.
It is yet another object of the invention to provide a method for the generation of lineage-restricted neuronal precursor cells from a CNS multipotent precursor cell able to generate both neurons and glia.
It is a still further object of the invention to provide pure differentiated populations of neuronal cells derived from lineage-restricted neuronal precursor cells.
It is still another object of the invention to provide methods of transfecting and transplanting such neuronal restricted precursor cells.
These and other objects can be achieved by providing an isolated, pure population of mammalian CNS neuron-restricted precursor cells. Preferably, such neuron-restricted precursor cells are capable of self-renewal, differentiation to CNS neuronal cells but not to CNS glial cells, and expressing embryonic neural cell adhesion molecule (E-NCAM), but not expressing a ganglioside recognized by A2B5 antibody. These neuron-restricted precursor cells may or may not express nestin or xcex2-III tubulin. Thus, embryonic neural cell adhesion molecule (E-NCAM) is a defining antigen for these cells. The NRP cells are able to differentiate into neurons that are capable of releasing and responding to neurotransmitters. These neurons can demonstrate receptors for these neurotransmitters, and such cells are capable of expressing neurotransmitter-synthesizing enzymes. The NRP cells are also capable of differentiating into neurons that can form functional synapses and/or develop electrical activity. The NRP cells are also capable of stably expressing at least one material selected from the group consisting of growth factors for such cells, differentiation factors for such cells, maturation factors for such cells, and combinations of any of these. Further, the present neuron-restricted precursor cells may be selected, chosen, and isolated from human primates, non-human primates, equines, canines, felines, bovines, porcines, ovines, lagomorphs, and rodents.
A method of isolating a pure population of mammalian CNS neuron-restricted precursor cells comprises the steps of:
(a) isolating a population of mammalian multipotent CNS stem cells capable of generating both neurons and glia;
(b) incubating the multipotent CNS stem cells in a medium configured for inducing such cells to begin differentiating;
(c) purifying from the differentiating cells a subpopulation of cells expressing a selected antigen defining neuron-restricted precursor cells; and
(d) incubating the purified subpopulation of cells in a medium configured for supporting adherent growth thereof.
A preferred selected antigen defining neuron-restricted precursor cells is embryonic neural cell adhesion molecule. Preferably, the step of purifying the NRP cells comprises a procedure selected from the group consisting of specific antibody capture, fluorescence activated cell sorting, and magnetic bead capture. Specific antibody capture is especially preferred. In a preferred embodiment, the mammalian multipotent CNS stem cells are neuroepithelial stem cells. A preferred procedure for isolating a population of CNS neuroepithelial stem cells comprises:
(a) removing a CNS tissue from a mammalian embryo at a stage of embryonic development after closure of the neural tube but prior to differentiation of cells in the neural tube;
(b) dissociating cells comprising the neural tube removed from the mammalian embryo;
(c) plating the dissociated cells in feeder-cell-independent culture on a substratum and in a medium configured for supporting adherent growth of the neuroepithelial stem cells comprising effective amounts of fibroblast growth factor and chick embryo extract; and
(d) incubating the plated cells at a temperature and in an atmosphere conducive to growth of the neuroepithelial stem cells.
Preferably, the mammalian embryo is selected from the group consisting of human and non-human primates, equines, canines, felines, bovines, porcines, ovines, lagomorphs, and rodents. It is also preferred that the substratum is selected from the group consisting of fibronectin, vitronectin, laminin, and RGD peptides. In a preferred embodiment, the medium comprises effective amounts of fibroblast growth factor and neurotrophin 3 (NT-3).
A method of isolating a pure population of mammalian CNS neuron-restricted precursor cells comprises the steps of:
(a) removing a sample of CNS tissue from a mammalian embryo at a stage of embryonic development after closure of the neural tube but prior to differentiation of glial and neuronal cells in the neural tube;
(b) dissociating cells comprising the sample of CNS tissue removed from the mammalian embryo;
(c) purifying from the dissociated cells a subpopulation expressing a selected antigen defining neuron-restricted precursor cells;
(d) plating the purified subpopulation of cells in feeder-cell-independent culture on a substratum and in a medium configured for supporting adherent growth of the neuron-restricted precursor cells; and
(e) incubating the plated cells at a temperature and in an atmosphere conducive to growth of the neuron-restricted precursor cells.
Preferably, the selected antigen defining neuron-restricted precursor cells is embryonic neural cell adhesion molecule. It is also preferred that the step of purifying comprises a procedure selected from the group consisting of specific antibody capture, fluorescence activated cells sorting, and magnetic bead capture. Specific antibody capture is especially preferred. It is further preferred that the mammalian embryo is selected from the group consisting of human and non-human primates, equines, canines, felines, bovines, porcines, ovines, lagomorphs, and rodents.
A method of obtaining postmitotic neurons comprises:
(a) providing neuron-restricted precursor cells and culturing the neuron-restricted precursor cells in proliferating conditions; and
(b) changing the culture conditions of the neuron-restricted precursor cells from proliferating conditions to differentiating condition, thereby causing the neuron-restricted precursor cells to differentiate into postmitotic neurons.
The changing of the culture conditions preferably comprises adding retinoic acid to basal medium or withdrawing a mitotic factor from basal medium. Such a mitotic factor is fibroblast growth factor. Changing the culture conditions can also comprise adding a neuronal maturation factor to basal medium. Preferred neuronal maturation factors are selected from the group consisting of sonic hedgehog, BMP-2, BMP-4, NT-3, NT-4, CNTF, LIF, retinoic acid, brain-derived neurotrophic factor (BDNF), and combinations of any of the above.
Another preferred embodiment of the invention comprises an isolated cellular composition comprising the mammalian CNS neuron-restricted cells described herein. Another preferred embodiment of the invention comprises a pharmaceutical composition comprising a therapeutically effective amount of such composition and a pharmaceutically acceptable carrier.
A method for treating a neuronal disorder in a mammal comprises administering to such mammal a therapeutically effective amount of the isolated cellular composition comprising the mammalian CNS neuron-restricted cells described herein. Another method for treating a neuronal disorder in a mammal comprising administering to said mammal a therapeutically effective amount of such pharmaceutical composition and a pharmaceutically acceptable carrier. Such composition can be administered by a route selected from the group consisting of intramuscular administration, intrathecal administration, intraperitoneal administration, intravenous administration, and combinations of any of the above. This method can also include the administration of a member selected from the group consisting of differentiation factors, growth factors, cell maturation factors and combinations of any of the above. Such differentiation factors are preferably selected from the group consisting of retinoic acid, BMP-2, BMP-4, and combinations of any of the above.
A method for treating neurodegenerative symptoms in a mammal comprises the steps of:
(a) providing a pure population of neuronal restricted precursor cells;
(b) genetically transforming such neuronal restricted precursor cells with a gene encoding a growth factor, neurotransmitter, neurotransmitter synthesizing enzyme, neuropeptide, neuropeptide synthesizing enzyme, or substance that provides protection against free-radical mediated damage thereby resulting in a transformed population of neuronal restricted precursor cells that express such growth factor, neurotransmitter, neurotransmitter synthesizing enzyme, neuropeptide, neuropeptide synthesizing enzyme, or substance that provides protection against free-radical mediated damage; and
(c) administering an effective amount of said transformed population of neuronal restricted precursor cells to such mammal.
A method or screening compounds for neurological activity comprising the steps of:
(a) providing a pure population of neuronal restricted precursor cells or derivatives thereof or mixtures thereof cultured in vitro;
(b) exposing such cells or derivatives thereof or mixtures thereof to a selected compound at varying dosages; and
(c) monitoring the reaction of such cells or derivatives thereof or mixtures thereof to said selected compound for selected time periods.
A method for treating a neurological or neurodegenerative disease comprises administering to a mammal in need of such treatment an effective amount of neuronal restricted precursor cells or derivatives thereof or mixtures thereof. Such neuronal restricted precursor cells or derivatives thereof or mixtures thereof can be from either a heterologous donor or an autologous donor. The donor can be a fetus, juvenile, or adult.
A method of isolating a pure population of mammalian CNS neuron-restricted precursor cells comprises the steps of:
(a) providing a sample of mammalian embryonic stem cells;
(b) purifying from the mammalian embryonic stem cells a subpopulation expressing a selected antigen defining neuron-restricted precursor cells;
(c) plating the purified subpopulation of cells in feeder-cell-independent culture on a substratum and in a medium configured for supporting adherent growth of the neuron-restricted precursor cells; and
(d) incubating the plated cells at a temperature and in an atmosphere conducive to growth of the neuron-restricted precursor cells.